EP2194147B1 - Multiplexverstärkung von Polynucleotiden - Google Patents

Multiplexverstärkung von Polynucleotiden Download PDF

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EP2194147B1
EP2194147B1 EP09016059.9A EP09016059A EP2194147B1 EP 2194147 B1 EP2194147 B1 EP 2194147B1 EP 09016059 A EP09016059 A EP 09016059A EP 2194147 B1 EP2194147 B1 EP 2194147B1
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amplification
multiplex
primer
primers
reaction
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EP2194147A1 (de
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Mark R. Andersen
David W. Ruff
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Applied Biosystems LLC
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Applied Biosystems LLC
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification

Definitions

  • the present invention relates to the field of molecular biology, and in particular provides kits for amplifying polynucleotide sequences of interest in a multiplex fashion. Once amplified, the multiplex amplification product can be used in downstream analyses without further purification or manipulation.
  • the relative concentrations of the various amplicons generated during the amplification can be maintained sufficiently for detecting changes in the relative concentrations by increasing the amount or concentration of DNA polymerase used and/or the length of the time or duration of the primer extension reaction per cycle.
  • multiplex amplifications carried out in the absence of such oligonucleotide probes can be divided into aliquots, with or without prior dilution, for subsequent analysis without further purification or manipulation.
  • Samples amplified in a multiplex fashion may be used in virtually any subsequent analysis or assay without further purification or manipulation.
  • the product of the multiplex amplification may be used for single polynucleotide polymorphism ("SNP") analysis, genotyping analysis, gene expression analysis, fingerprinting analysis, analysis of gene mutations for genetic diagnoses, analysis of rare expressed genes in cells, nucleic acid sequencing (e.g., U.S. Patent No. 6,428,986 ), nucleic acid mini-sequencing (e.g., U.S. Patent No. 6,479,242 ), and for hybridizing to arrays (e.g., U.S. Patent No. 6,485,944 ).
  • SNP single polynucleotide polymorphism
  • the kit may further include oligonucleotide probes useful for SNP detection or analysis, oligonucleotide microarrays, such as microarrays suitable for gene expression or SNP analyses, and/or "tailed" primers (see, e.g., Bengra et al., 2002, Clin. Chem. 48:2131-2140 ; Myakishev et al., 2001, Genome Res. 11:163-169 ; and U.S. Patent No. 6,395,486 ) for universal amplification, detection and/or purification.
  • the kit further includes reagents suitable for carrying out a plurality of single-plex quantitative or real-time amplification reactions.
  • Such reagents typically include a set of quantitative or real-time amplification primers, an oligonucleotide probe labeled with a labeling system suitable for monitoring the quantitative real-time amplification reaction, a DNA polymerase at a concentration suitable for single-plex amplification and/or mixtures of dNTPs suitable for template-dependent DNA synthesis.
  • the kit may include one or more of any of these additional reagents.
  • each amplification primer pair includes two amplification primers, one forward amplification primer and one reverse amplification primer, as is well-known in the art.
  • the amplification primer pairs may be sequence-specific and may be designed to hybridize to sequences that flank a sequence of interest to be amplified.
  • the actual nucleotide sequences of each primer pair may depend upon the sequence of interest to be amplified, and will be apparent to those of skill in the art.
  • the multiplex amplification reactions may be carried out with a variety of different DNA polymerases (or mixture of DNA polymerases), but are preferably carried out in the presence of one or more thermostable polymerases. Suitable thermostable polymerases include, but are not limited to, Taq and Tth (commercially available from Applied Biosystems, an Applera Corporation business). Moreover, like conventional RT-PCR amplification reactions, multiplex RT-PCR amplification reactions may be carried out with a variety of different reverse transcriptases (or mixture of reverse transcriptases), although in some embodiments thermostable reverse-transcriptions are preferred.
  • the multiplex amplifications may be carried out with commercially-available amplification reagents, such as, for example, AmpliTaq® Gold PCR Master Mix, TaqMan® Universal Master Mix and TaqMan® Universal Master Mix No AmpErase® UNG, all of which are available commercially from Applied Biosystems, an Applera Corporation business.
  • commercially-available amplification reagents such as, for example, AmpliTaq® Gold PCR Master Mix, TaqMan® Universal Master Mix and TaqMan® Universal Master Mix No AmpErase® UNG, all of which are available commercially from Applied Biosystems, an Applera Corporation business.
  • a multiplex reaction is 100% efficient generally when the amounts of each of the amplicons produced in the multiplex amplification are identical to the amounts of the respective amplicons produced in individual, single-plex conventional PCR or RT-PCR reactions with the same target polynucleotides. Multiplex amplifications are considered "highly efficient" for a specific amplicon when the amount of the specified amplicon generated in a multiplex reaction is within about ⁇ 90% of the amount generated in a single-plex, conventional PCR or RT-PCR reaction.
  • the observed efficiency of a multiplex amplification reaction can be assessed using real-time PCR methods.
  • the observed efficiency of amplification of a specific target sequence can be determined by amplifying a plurality of polynucleotides, which includes the specific target, in a multiplex PCR amplification reaction using a plurality of primer sets, each of which is suitable for amplifying a different target sequence of interest.
  • the multiplex amplification is carried out for N thermal cycle steps, where N can be selected by the user.
  • Any number of specific target sequences in a multiplex amplification can be similarly analyzed.
  • a user can select a selection criteria (i.e. a "cut-off" value) for the observed efficiency such as, for example, 50%, 70%, 80%, 90%, 95% or 99%.
  • a selection criteria i.e. a "cut-off" value
  • the observed efficiencies for each primer set in a multiplex amplification can be determined and the primers grouped according to whether their observed efficiencies equal or exceed the selection criteria.
  • Primer sets that do not meet or exceed the selection criteria can be analyzed individually in singleplex amplifications, or can be re-grouped into one or more separate pools of primer sets for further analysis.
  • PCR and RT-PCR reactions can be broken up into three phases: an exponential phase in which the amount of amplicon accumulates exponentially every cycle (i.e., doubles every cycle); a linear phase in which the amount of amplicon accumulates at a variable rate every cycle (i.e., the reaction begins to slow); and a plateau phase, where the reaction has stopped, no more amplicon is being produced and, if left long enough, the amplicon will begin to degrade.
  • the multiplex amplification is carried out for as many as 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more cycles. In specific embodiments, the multiplex amplification is carried out for 2-12 cycles, inclusive, for 5-11 cycles, inclusive, or for up to 14 cycles (e.g., see Example 7). A high number of cycles may be required in certain applications where a large plurality of downstream assays are to be performed.
  • the product of the multiplex amplification may be used in subsequent amplification reactions, such as the quantitative or real-time amplification assays commonly used for gene expression analysis.
  • quantitative or real-time amplification assays total RNA from a sample is amplified by RT-PCR in the presence of amplification primers suitable for specifically amplifying a specified gene sequence of interest and an oligonucleotide probe labeled with a labeling system that permits monitoring, for example via the 5'-exonuclease activity of the DNA polymerase employed in the RT-PCR amplification, of the quantity of amplicon that accumulates in the amplification reaction in real-time.
  • 5'-exonuclease gene quantification assays require relatively large amounts of starting RNA (e.g. , from 1-10 ⁇ g). Owing to this large sample requirement, 5'exonuclease gene quantification assays and other gene quantification assays have not been suitable for detecting genes expressed at low copy numbers, or in instances where only limited quantities of sample is available ( e.g. , from clinical biopsies, etc.).
  • the multiplex amplification reactions described herein are ideal for use in connection with the such downstream gene expression analyses, such as, for example the 5'-exonuclease gene quantification assay.
  • Polynucleotides present in samples at extremely low copy numbers, and/or samples obtained from a few or even a single cell, may be multiplex amplified so as to provide amounts of sample suitable for tens, hundreds or even thousands of quantitative or real-time amplification assays.
  • the product of the multiplex amplification reaction is divided, either with or without prior dilution, amongst a plurality of single-plex quantitative or real-time amplification reactions.
  • Each single-plex quantitative or real-time amplification reaction is carried out in a conventional manner with a single set of amplification primers and a suitable probe.
  • the amplification primer pair or set used for the single-plex quantitative or real-time amplification can be the same as one of the primer pairs or sets used in the multiplex amplification reaction.
  • the multiplex amplification product can be used directly in such subsequent single-plex amplifications without further purification or manipulation.
  • the various enzymes, dNTPs, amplification primers and other optional reagents carried over from the multiplex amplification do not interfere with the accuracy of the subsequent quantitative or real-time amplification assays.
  • the present inventor has surprisingly discovered, in certain embodiments, that the multiplex amplification substantially maintains the copy number ratios, presumably due to high efficiency of amplification, so that the copy numbers or expression levels of the original sample can be ascertained from the multiplex amplified sample.
  • relative copy numbers from the original sample can thus be determined and used, for example, in various downstream applications, such as gene expression studies.
  • Samples amplified in a multiplex fashion may be used in a wide variety of subsequent analysis or assay without further purification or manipulation.
  • the product of the multiplex amplification may be used for single polynucleotide polymorphism ("SNP") analysis, genotyping analysis, gene expression analysis, fingerprinting analysis, analysis of gene mutations for genetic diagnoses, analysis of rare expressed genes in cells, nucleic acid sequencing (e.g., U.S. Patent No. 6,428,986 ), and nucleic acid mini-sequencing (e.g., U.S. Patent No. 6,479,242 ).
  • SNP single polynucleotide polymorphism
  • the detected signal will depend upon the particular method utilized. For example, when using real-time PCR, the detected signal can be related to a fluorescence intensity. Amplification products can be separated and detected by any of a variety of techniques known to those of skill in the art (see, e.g., U.S. Patent No. 6,618,679 ). The data obtained from the detection can be stored and analyzed to obtain a set of gene expression data. When using a microfabricated DNA array, the detected signal can be a hybridization intensity.
  • the product of a multiplex amplification can be applied to solid supports containing polynucleotide hybridization probes for differentially expressed genes.
  • Any solid surface to which polynucleotides can be bound, either directly or indirectly, either covalently or non-covalently, can be used.
  • Non-limiting examples of such supports include filters, polyvinyl chloride dishes, beads, glass slides etc.
  • a particular example of a solid support is a high density array or DNA chip. These contain a particular hybridization probe in a predetermined location on the array. In some embodiments, each predetermined location may contain more than one molecule of the probe, but each molecule within the predetermined location has an identical sequence. Such predetermined locations are termed features.
  • Hybridization probe arrays for expression monitoring can be made and used according to any techniques known in the art. (See for example, Lockhart, D. J. et al., 1996, Nature Biotechnology 14:1675-1680 ; McGall, G. et al., 1996, Proc. Nat. Acad. Sci. USA 93:13555-13460 ; and U.S. Patent Nos. 6,033,860 , 6,309,822 , 6,485,944 , and 6,548,257 ).
  • the present inventor has surprisingly discovered that the presence of conventional concentrations of oligonucleotide probes, such as 5'-exonuclease probes, in a multiplex amplification reaction does not interfere with the performance or efficiency of the multiplex amplification. Nor does the presence of such probes interfere with downstream analyses, such as single-plex quantitative or real-time amplification assays or other analyses, carried out with the product of the multiplex amplification.
  • This discovery permits multiplex amplifications to be carried out using commercially-available, off-the-shelf quantitative or real-time amplification reagents, such as the Assays-On-Demand reagents commercially available from Applied Biosystems (an Applera Corporation business).
  • kits suitable for carrying out a multiplex amplification followed by a plurality of single-plex quantitative or real-time amplification assays can be readily created from readily-available 5'-exonuclease reagents without requiring additional manipulations or purification.
  • the primers for performing the multiplex amplification can be created by pooling together 5'-exonuclease reagents comprising a pair of amplification primers and a 5'-exonuclease probe.
  • 5'-exonuclease probes in the multiplex amplification reaction and subsequent single-plex 5'-exonuclease amplification assays does not interfere with either amplification.
  • a plurality of 5'-exonuclease amplification primer/probe sets are selected by the user and pooled together to yield a plurality of amplification primer pairs or sets suitable for multiplex amplification (the pool also includes the plurality of 5'-exonuclease probes).
  • the 5'-exonuclease primer/probe sets may be dispensed among the wells of a micro fluidic card that can be used directly on an instrument designed for quantitative or real-time amplification analysis, such as the AB Prism 7900 HT instrument available from Applied Biosystems (an Applera Corporation business).
  • a suitable microcard is described in U.S. Patent No. 6,126,899 and a commercial embodiment is the 7900HT Micro Fluidic card available from Applied Biosystems (an Applera Corporation business).
  • oligonucleotide probes may be DNA, RNA, PNA, LNA or chimeras composed of one or more combinations thereof.
  • the oligonucleotides may be composed of standard or non-standard nucleobases or mixtures thereof and may include one or more modified interlinkages, as previously described in connection with the amplification primers.
  • the oligonucleotide probes may be suitable for a variety of purposes, such as, for example to monitor the amount of an amplicon produced, to detect single nucleotide polymorphisms, or other applications as are well-known in the art.
  • oligonucleotide probes can vary broadly, and in some embodiments can range from as few as two as many as tens or hundreds of nucleotides, depending upon the particular application for which the probe was designed. In one specific embodiment, the oligonucleotide probes range in length from about 15 to 35 nucleotides. In another specific embodiment, the oligonucleotide probes range in length from about 15 to 25 nucleotides. In yet another specific embodiment, oligonucleotide probes can range from 25 to 75 nucleotides. In other embodiments, the probes range in length from about 6 to 75 nucleotides or from about 12 to 22 nucleotides.
  • An oligonucleotide probe can include a 5' tag portion for binding with a mobility modifier (e.g., as described in U.S. Patent No. 6,395,486 ).
  • oligonucleotide probes present in a multiplex amplification are suitable for monitoring the amount of amplicon(s) produced as a function of time.
  • oligonucleotide probes include, but are not limited to, the 5'-exonuclease assay (TaqMan®) probes described above (see also U.S. Patent No. 5,538,848 ), various stem-loop molecular beacons (see, e.g., U.S. Patent Nos.
  • peptide nucleic acid (PNA) light-up probes self-assembled nanoparticle probes
  • ferrocene-modified probes described, for example, in U.S. Patent No. 6,485,901 ; Mhlanga et al., 2001, Methods 25:463-471 ; Whitcombe et al., 1999, Nat. Biotechnol. 17:804-807 ; Isacsson et al., 2000, Mol. Cell. Probes. 14:321-328 ; Svanvik et al., 2000, Anal Biochem.
  • Performing multiplex amplification in the presence of these various oligonucleotide probes permits a great deal of flexibility in designing or creating sets of amplification primers for multiplex amplifications.
  • Commercially available primer sets including such oligonucleotide probes can be simply pooled together without prior removal of the oligonucleotide probes and used for multiplex amplification without further manipulation.
  • the oligonucleotide probes can be removed from pooled primer sets prior to multiplex amplification. Such removal can be effected using pairs of specific binding molecules, such as biotin/avidin or antibody/antigen.
  • a biotin-labeled oligonucleotide probe can be removed by avidin binding.
  • labeled nucleotide probes can be photobleached using laser or other light sources.
  • the multiplex amplification may also be carried out in the presence of dye molecules suitable for, for example, monitoring the accumulation of amplification products at the end of the amplification and/or during the amplification as a function of time.
  • dyes include dyes that produce a detectable signal, such a fluorescence, when bound to double-stranded polynucleotides.
  • suitable dyes include common nucleic acid stains, such as intercalating dyes and minor groove binding dyes, as are well-known in the art.
  • the dye is SYBR® Green I or II, ethidium bromide, or YO-PRO-1 (available from Molecular Probes, Eugene, OR).
  • Such dyes can be used at conventional concentrations commonly employed in the art (see, e.g., U.S. Patent No. 5,994,056 ).
  • a multiplex amplification in the presence of such a dye molecule, or in the presence of suitable oligonucleotide probes, Applicants have discovered a general method for characterizing pooled sets of primers.
  • the amplification is monitored in real time, and a cycle threshold value ("Ct pool ”) obtained.
  • This is an additive signal produced by the summation of all of the amplicons.
  • oligonucleotide probes are used for the real time multiplex amplification, a separate probe is present for each target sequence being amplified, and all of the probes use the same signaling system.
  • the method is especially useful in providing a rapid and convenient test of pooled reagents that may be provided in ready-made, pre-optimized, kits.
  • a pool of reagents can be prepared by mixing commercially available primer sets such as the Assays-on-DemandTM Gene Expression products or the primer sets available in the QuantiTect Gene Expression Assays (Qiagen), as further described herein.
  • the relative levels of the various polynucleotides in a sample can be determined and compared to a reference sequence (i.e., normalized).
  • reference sequence refers to a nucleic acid sequence serving as a target of amplification in a sample that provides a control for the assay.
  • the reference may be internal (or endogenous) to the sample source, or it may be an externally added (or exogenous) to the sample.
  • a reference sequence is typically amplified during the multiplex amplification. For example, when performing gene expression analysis, at least one amplification target in a multiplex set that is endogenous to the sample can be selected as a reference sequence.
  • This reference can be a target that has been independently shown to exhibit a fairly constant expression level (for example, a "housekeeping" gene). Examples of such housekeeping genes include GAPDH, ⁇ -actin, 18SRNA and cyclophilin.
  • the Ct value from an endogenous reference sequence can provide a control for converting Ct values of other target sequences into relative expression levels.
  • a plurality of control targets/reference sequences that have relatively constant expression levels may be included in the multiplexed amplification to serve as controls for each other.
  • the reference sequence can be an external reference sequence.
  • an external reference sequence may be a defined quantity of either RNA, added to the sample prior to reverse transcription, or DNA (e.g., cDNA), added prior to the multiplex amplification.
  • a multiplex polymerase chain reaction amplification is carried out to amplify a sample, such as an RNA or a cDNA sample, using a plurality of primer sets for amplifying target sequences, and including a primer set for amplifying a reference sequence.
  • a Ct target value is obtained for each target sequence and a Ct ref value is obtained for the reference sequence.
  • the Ct ref value is subtracted from each Ct target value to yield a ⁇ Ct target value for each target sequence.
  • the Ct pool value (as described above) can be used as the reference value.
  • the Ct pool value can be obtained and subtracted from each Ct target value (i.e., the Ct pool value is used in place of the Ct ref value) to yield a ⁇ Ct target value.
  • Certain applications of the present methods concern analyzing samples obtained from a cell, cell line, tissue or organism that has undergone a treatment. For example, up-regulation or down-regulation of certain genes can be analyzed.
  • treatment refers to the process of subjecting one or more cells, cell lines, tissues, or organisms to a condition, substance, or agent (or combination thereof) that may cause the cell, cell line, tissue or organism to alter its gene expression profile.
  • a treatment may include a range of chemical concentrations and exposure times, and replicate samples may be generated.
  • untreated control refers to a sample obtained from a cell, cell line, tissue or organism that has not been exposed to a treatment.
  • mRNA (or cDNA) from an untreated control can be amplified in a multiplex amplification in the same manner as a sample from a treated cell, cell line, tissue or organism.
  • Cycle threshold values obtained from both a treated sample and from an untreated control can be normalized, as described above.
  • a cycle threshold value for the untreated control (“Ct untreated ") can be obtained for each target sequence in the untreated control sample and a Ct ref obtained for each reference sequence, as described above. The Ct ref is subtracted from each Ct untreated value to obtain ⁇ Ct unreated values.
  • a passive reference containing a fluorescent dye e.g. ROX
  • a fluorescent dye e.g. ROX
  • primer sets are pooled, and used in a multiplex amplification in the presence of a reagent suitable for monitoring the amplification as a function of time.
  • a reagent suitable for monitoring the amplification as a function of time.
  • a reagent include oligonucleotide probes.
  • Other examples include dye molecules (e.g. intercalating dyes and minor groove binding dyes).
  • reagents and kits suitable for carrying out the multiplex amplification and various two-step reactions and/or assays described herein may be modeled after reagents and kits suitable for carrying out conventional PCR and RT-PCR amplification reactions, with the exception that instead of a single set of amplification primers, the reagents and/or kits include a plurality of amplification primers packaged in a single container, wherein the single container may additionally contain one or more oligonucleotide probes, as described herein.
  • kits may optionally include reagents packaged for downstream or subsequent analysis of the multiplex amplification product.
  • the kit includes a container comprising a plurality of amplification primer pairs or sets, each of which is suitable for amplifying a different sequence of interest, and a plurality of reaction vessels, each of which includes a single set of amplification primers suitable for amplifying a sequence of interest.
  • the primers included in the individual reaction vessels can, independently of one another, be the same or different as a set of primers comprising the plurality of multiplex amplification primers.
  • both the container and plurality of reaction vessels further include 5'-exonuclease probes such that the kit is suitable for carrying out the multistep assay illustrated in FIG. 2 .
  • the plurality of reaction vessels is a multiwell plate.
  • the amplification primer mix for the 95-plex amplification was prepared by pooling 10 ⁇ L from each of 95 different randomly selected 20X Assays-on-DemandTM Gene Expression Products (Applied Biosystems, an Applera Corporation business, Catalog Nos.
  • Each 20X Assays-on-DemandTM Gene Expression Product contained two unlabeled amplification primers (18 ⁇ M each primer) and one FAM-labeled TaqMan® MGB probe (5 ⁇ M).
  • 95-Plex amplifications were carried out with this amplification primer mix using DNA polymerase concentrations ranging from 1 Unit per 20 ⁇ L reaction volume (1 U/20 ⁇ L) to 17 U/20 ⁇ L.
  • DNA polymerase concentrations ranging from 1 Unit per 20 ⁇ L reaction volume (1 U/20 ⁇ L) to 17 U/20 ⁇ L.
  • 5 ⁇ L pooled primer mix 10 ⁇ L 2X TaqMan® Universal PCR Master Mix ("2X Master Mix”; Applied Biosystems, an Applera Corporation business, Cat.
  • 2X Master Mix comprises AmpliTaq Gold® DNA polymerase (0.1 U/ ⁇ L), AmpErase® UNG, dNTPs with dUTP, a passive reference and optimized buffer components. 95-Plex amplifications carried out at higher DNA polymerase concentrations were prepared by spiking the reaction with the appropriate amount of AmpliTaq Gold® (5 U/ ⁇ l; Applied Biosystems Catalog No. N808024).
  • each 95-plex amplification was diluted to 200 ⁇ l with water (10-fold) and divided for 95 individual single-plex real-time amplification reactions.
  • Each single-plex amplification used as primers/probes one of the 20X Assays-on-DemandTM Gene Expression Products described above, with a different set of primers per reaction.
  • the following volumes of reagents were used for the single-plex real-time amplifications: 2 ⁇ L diluted 95-plex amplification product, 1 ⁇ L 20X Assays-on-DemandTM Gene Expression Product, 10 ⁇ L 2X Master Mix and water to yield a 20 ⁇ L reaction volume.
  • the performance of the 95-plex amplification increased with increasing DNA polymerase concentration over a range of 1-5 U/20 ⁇ L reaction volume, at which concentration the performance plateaued prior to decreasing slightly. From this experiment, it was determined that the optimal spiked DNA polymerase concentration for carrying out multiplex amplifications using the reaction conditions described above is in the range of 4-6 Units per every 20 ⁇ L reaction volume. The decrease in performance observed at higher levels of spiked DNA polymerase is believed to have been caused by exceedingly high concentrations of components of the enzyme storage buffer, e.g. glycerol, in the multiplex amplification reaction.
  • the optimal spiked DNA polymerase concentration for carrying out multiplex amplifications using the reaction conditions described above is in the range of 4-6 Units per every 20 ⁇ L reaction volume.
  • the decrease in performance observed at higher levels of spiked DNA polymerase is believed to have been caused by exceedingly high concentrations of components of the enzyme storage buffer, e.g. glycerol, in the multiplex amplification reaction.
  • Another significant advantage of multiplex amplifications is the ability to carry out the reaction in the presence of oligonucleotide probes without significant interference during either the multiplex amplification or downstream amplifications carried out on the multiplex amplification product.
  • This former advantage is apparent from Example 2, supra .
  • efficient amplification was achieved in the multiplex amplification step, which by virtue of utilizing Assays-On-Demand TM reagents to create the multiplex primer pool, included TaqMan® MGB oligonucleotide probes in the reaction.
  • a single-plex RNase P assay (DNA specific) was run with 1 ng of genomic DNA in the presence or absence of a 5X concentration of the 95-plex primer/probe pool (RNA-cDNA specific) described in Example 1.
  • the 5X concentration of 95-plex primer/probe pool was added to determine what effect it would have on the single-plex RNase assay.
  • the average Ct values of the two reactions are illustrated in FIG. 5 .
  • the presence of the 95-plex primer/probe pool did not affect the Ct value of the RNase P amplicon, demonstrating that single-plex RNase P assays can be carried out with the product of a multiplex amplification reaction without having to first remove the multiplex primers and/or probes.
  • the presence of the multiplex primers and/or probes does not deleteriously affect the performance of the single-plex RNase P assay.
  • multiplex amplifications permit downstream analysis of quantities of sample that would otherwise be too small for the desired type and/or number of analyses
  • multiplex amplifications were carried out with varying concentrations of sample cDNA ranging from 100 ng to 100 pg (approximately equivalent to a sample size of 5 cells).
  • concentration of cDNA was subjected to 95-plex amplification followed by 95 individual real-time amplification analyses as described in Example 1.
  • the average Ct values of the 95-plex amplifications as a function of sample cDNA concentration are provided in FIG. 6 .
  • there is a linear relationship between the sample cDNA concentration and average Ct value demonstrating that a large percentage (approx.
  • the target sequences amplified efficiently even though they were amplified simultaneously in a multiplexed fashion.
  • the level of sensitivity achieved demonstrates that samples from as few as 1-2 cells can be analyzed by real-time PCR following multiplex amplification.
  • the multiplex amplification yields a quantity of amplified sample sufficient for numerous downstream real-time PCR assays.
  • 186-plex, 369-plex, 738-plex and 1013-plex amplifications were carried out in four individual multiplex amplification reactions.
  • the amplification primer mix for each of the amplifications was prepared by pooling equal volumes of 186, 369, 738 or 1013 different randomly selected 20X Assays-on-DemandTM Gene Expression Products into four separate microcentrifuge tubes, respectively. For each of the four tubes, the pooled solution was dried using a SpeedVac® concentrator (Thermo Savant, Holbrook, NY).
  • the residue was re-suspended in deionized water such that the multiplexed amplification primers were at a 4X stock concentration (180 nM each primer) relative to the 1X working amplification primer concentration of 45nM.
  • the combined primers were present in the re-suspension at a concentration of 45.6 ⁇ M, and the FAM-labeled TaqMan MGB probes were present at 10.1 ⁇ M.
  • each of the above randomly selected 20X Assays-on-Demand Gene Expression Products was distributed into a series of 96-well plates (designated alphabetically plates IAA through IAO).
  • Each 20X Assays-on-Demand Gene Expression Product contained two unlabeled amplification primers (18 ⁇ M each primer) and one FAM-labeled TaqMan® MGB probe (5 ⁇ M).
  • Each of the amplifications (from the 186-, 369-, 738- or 1013-plex pooled primer mixtures) were carried out in a final volume of 50 ⁇ L, with the constituents being 12.5 ⁇ L of 4X pooled and re-suspended primer mix, 25 ⁇ L 2X TaqMan® Universal PCR Master Mix ("2X Master Mix"; Cat. #4324016 containing no UNG enzyme), 10 ⁇ L template cDNA (from a cDNA library; 25 ng total cDNA) and 2.5 ⁇ L AmpliTaq Gold® DNA polymerase (5U/ ⁇ L).
  • the 2X Master Mix included AmpliTaq Gold® DNA polymerase (0.1 U/ ⁇ L), dNTPs, a passive reference and optimized buffer components. Each of the four reactions were carried out for a total of 10 cycles (15 sec. melt at 95 °C; 4 min. anneal/extend at 60 °C) on an ABI Prism® 7700 instrument.
  • the product of each amplification was diluted with water and aliquoted for single-plex analysis. In the case of the 186- and 369-plex reactions, the product was diluted 1:5 prior to setting up the single-plex assays. For the 738- and 1013-plex amplifications, the product was diluted 1:10 prior to setting up the single-plex assays.
  • Each of these single-plex amplifications used as primers/probes one of the 20X Assays-on-DemandTM Gene Expression Products used in the multiplex amplification described above. and were distributed into a series of 96-well plates for liquid transfer convenience (designated alphabetically plates IAA through IAO). A different set of primer/probes was used in each single-plex reaction.
  • the following volumes of reagents were used for the single-plex ("assay") amplifications: 2.5 ⁇ L diluted 186-, 369-, 738- or 1013-plex amplification product, 0.5 ⁇ L 20X Assays-on-Demand Gene Expression Product, 5 ⁇ L 2X Master Mix and water to yield a 10 ⁇ L reaction volume. All assay amplifications were carried out for a total of 40 cycles (15 sec. melt at 95 °C; 1 min. anneal/extend at 60 °C) on an ABI Prism® 7900 instrument. The accumulation of amplicon was monitored in real time.
  • a method for preventing "carry over" contamination in PCR includes the use of dUTP in place of dTTP in the PCR mixture, followed by treatment of all subsequent PCR mixtures with uracil N-glycosylase (UNG) ( U.S. Patent 5,035,996 ).
  • UNG uracil N-glycosylase
  • a first 186-plex amplification was carried out as described in Example 5, but using TaqMan® Universal Master Mix, No AmpErase® UNG (Cat. #4324018), instead of Universal Master Mix (Cat. #4304437).
  • the multiplex amplification was extended for 14 cycles, instead of 10 cycles, as in Example 5.
  • the samples were chilled on ice after the amplification, and then subjected to single-plex PCR as described in Example 5.
  • UNG(+) Another 186-plex amplification (UNG(+)) was carried out as described in Example 5, but using Universal Master Mix (Cat. #4304437) (with UNG), except that the 186-plex amplification was extended through 14 cycles, and the samples were chilled on ice for 4 hours. The samples were subjected to single-plex PCR as described in Example 5.
  • Example 5 the effect of the presence of UNG in a multiplex amplification, carried out in a procedure similar to that described in Example 5, was evaluated.
  • the presence of UNG in the multiplex amplification had essentially no affect on the efficiency of amplification in the single-plex amplification step as compared to the UNG(-) sample.
  • Example 5 the multiplex amplification was carried out for 10 cycles. In the present Example, multiplex amplifications were carried out for 10, 12 and 14 cycles. Higher cycle numbers can increase the concentration of the amplification product, which allows a greater number of downstream assays, such as a greater number of single-plex amplifications, to be performed.
  • the average ⁇ Ct value increased as the number of thermal cycles increased. The standard deviations were essentially unchanged. These results indicate that there is no decrease in performance in going from 10 to 14 cycles. If amplifications are 100% efficient, then the ⁇ Ct between amplified and "mock" reactions will be 10 with 10 cycles of amplification, 12 with 12 cycles, and 14 with 14 cycles. In this Example, the average ⁇ Ct values approximated 10 (9.99), 12 (11.8), and 14 (14.13), respectively.
  • RNA in plasma can allow for early detection of disease states such as cancer, coronary and autoimmune dysfunctions and can also be used to monitor the success of drug treatment regimes by following gene expression.
  • This Example demonstrates generation of cDNA from a sample of mRNA followed by multiplex PCR amplification of the cDNA in the presence of a plurality of selected PCR primers within a single reaction mixture, with subsequent single-plex real-time PCR in the presence of each of the selected PCR primers.
  • Equal volumes (10 ⁇ l each) of 108 selected 20X Assays-on-Demand Gene Expression Products were pooled and dried down using a SpeedVac® concentrator (Thermo Savant, Holbrook, NY). The residue was re-suspended in deionized water to yield a primer concentration of 180 nM for each primer.
  • the 108 Assay-on-Demand primers corresponded to solid tissue and leukocyte specific amplicons, examples of which included: pinin, hexokinase-1, VEGF ⁇ , PRKCB1, LGALS3BP, cyclophilin A, GAS2L1, DDX1, TERT, BMPR2, LANCL1, and CCL5.
  • the final volume was 250 ⁇ l (240 ng plasma RNA), and 50 ⁇ l was aliquoted into separate wells of a 96-well plate.
  • the reverse transcription reaction was carried out for 30 min at 48°C, followed by denaturation for 10 min at 95°C.
  • the multiplex amplification was carried out for a total of 14 cycles (each cycle: 15 sec at 95°C; 4 min. anneal/extend at 60°C). These reactions were carried out on an ABI Prism® 7700 instrument.
  • the results indicate that DNA obtained from reverse transcription of human plasma RNA, and subjected to multiplex amplification in the presence of a plurality of primer pairs, can subsequently be single-plex amplified using the same primer pairs without optimization.
  • step (i) is further carried out in the presence of a reverse transcriptase such that the polymerase chain reaction is reverse-transcription polymerase chain reaction and wherein the one or more cDNA molecules is obtained from mRNA derived from the sample.
  • the one or more cDNA molecules comprise a cDNA library.
  • said quantifying comprises analysis by a method selected from at least one of the group consisting of real-time polymerase chain reaction amplification, DNA microarray hybridization analysis, electrophoresis and chromatography.
  • step (i) Disclosed is also the above method in which the polymerase chain reaction of step (i) is carried out for a number of cycles such that the amplification remains in the linear range.
  • step (i) Disclosed is also the above method in which the amplification in step (i) is achieved with a thermostable DNA polymerase.
  • At least one oligonucleotide probe is labeled with a moiety capable of producing a detectable signal.
  • the label is a fluorophore.
  • said at least one oligonucleotide probe is selected from the group consisting of 5'-exonuclease probes, stem-loop beacon probes and stemless beacon probes.
  • said at least one oligonucleotide probe comprises a plurality of oligonucleotide probes, each of which is complementary to a region of a different amplified target gene sequence of interest.
  • the product of step (i) is divided into a plurality of aliquots and said quantifying in step (ii) is performed on said aliquots.
  • step (ii) comprises amplifying the product in each aliquot by polymerase chain reaction in the presence of an amplification primer set suitable for amplifying one of the target sequences of the plurality.
  • step (ii) is further carried out in the presence of an oligonucleotide probe complementary to a region of a different amplified target gene sequence of interest, wherein each probe in step (ii) comprises one of the oligonucleotide probes in step (i).
  • step (i) Disclosed is also the above method in which the sequences of the amplification primer sets of step (i) are the same as the sequences of the amplification primer sets of step (ii). Disclosed is also the above method in which the amplifying in step (ii) is further carried out in the presence of a molecule that produces a detectable signal when bound to a double-stranded polynucleotide suitable for monitoring the amplification reaction as a function of time. Disclosed is also the above method in which the molecule is selected from the group consisting of an intercalating dye and a minor groove binding dye. Disclosed is also the above method in which the molecule is selected from the group consisting of SYBR (S green I and ethidium bromide.
  • SYBR S green I and ethidium bromide.
  • a method for determining a gene expression profile in a sample comprising the steps of : (i) amplifying one or more cDNA molecules derived from said sample by polymerase chain reaction in the presence of a plurality of amplification primer sets suitable for amplifying target gene sequences of interest; (ii) identifying amplified target gene sequences having an observed efficiency of amplification greater than a selected level; and (iii) quantifying the target gene sequences identified in step (ii) to obtain a gene expression profile.
  • step (i) is further carried out in the presence of a reverse transcriptase such that the polymerase chain reaction is reverse-transcription polymerase chain reaction and wherein the one or more cDNA molecules is obtained from mRNA derived from the sample.
  • said quantifying comprises analysis by a method selected from at least one of the group consisting of real-time polymerase chain reaction amplification, DNA microarray hybridization analysis, electrophoresis and chromatography.
  • step (i) Disclosed is also the above method in which the amplifying in step (i) is further carried out in the presence of an oligonucleotide probe complementary to a region of an amplified target gene sequence of interest, said probe being labeled with a labeling system suitable for monitoring the amplification reaction in step (i) as a function of time.
  • step (i) is divided into a plurality of aliquots and said quantifying in step (ii) is performed on said aliquots.
  • step (ii) comprises amplifying the product in one or more separate aliquots by polymerase chain reaction in the presence of an amplification primer set suitable for amplifying one of the target sequences of the plurality.
  • step (ii) is further carried out in the presence of a molecule that produces a detectable signal when bound to a double-stranded polynucleotide suitable for monitoring the amplification reaction as a function of time.
  • the molecule is selected from the group consisting of an intercalating dye and a minor groove binding dye.
  • step (i) The above method in which the polymerase chain reaction of step (i) is carried out for a number of cycles such that the amplification remains in the linear range.
  • a method of generating a plurality of target sequences of interest comprising the step of: amplifying by polymerase chain reaction one or more target polynucleotides in the presence of a plurality of amplification primers suitable for amplifying target sequences of interest and in the presence of at least one oligonucleotide probe complementary to a region of an amplified target sequence of interest, said oligonucleotide probe being optionally labeled with a labeling system suitable for monitoring an amplification reaction as a function of time.
  • said at least one oligonucleotide probe comprises a plurality of oligonucleotide probes, each of which is complementary to a region of an amplified target sequence of interest.
  • the product of the amplification is further subjected to at least one assay selected from the group consisting of single polynucleotide polymorphism analysis, genotyping analysis, gene expression analysis, fingerprinting analysis, analysis of gene mutations for genetic diagnoses, analysis of rare expressed genes in cells, nucleic acid sequencing, nucleic acid mini-sequencing and gene expression analysis.
  • at least one assay selected from the group consisting of single polynucleotide polymorphism analysis, genotyping analysis, gene expression analysis, fingerprinting analysis, analysis of gene mutations for genetic diagnoses, analysis of rare expressed genes in cells, nucleic acid sequencing, nucleic acid mini-sequencing and gene expression analysis.
  • the product of the amplification is further subjected to at least one assay selected from the group consisting of chromatography, electrophoresis, and staining with a dye or hybridization probe.
  • a method of generating a plurality of different target sequences of interest comprising the step of:
  • the molecule is selected from the group consisting of an intercalating dye and a minor groove binding dye.

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  1. Kit zur Ausführung von Multiplex-Amplifikationen, umfassend mehrere Amplifikationsprimersätze, die zur Ausführung einer Multiplex-Amplifikation geeignet sind, und ein oder mehrere einzelsträngige Oligonukleotidproben, die zu allen oder einem Teil einer amplifizierten Zielsequenz komplementär sind, die in einem einzigen Behälter verpackt ist, und
    mehrere Reaktionsgefäße, von denen jedes einen einzigen Satz von Amplifikationsprimern umfasst, die zum Amplifizieren einer Sequenz von Interesse geeignet sind.
  2. Kit nach Anspruch 1, ferner umfassend ein DNA-Polymeraseenzym, ein Reverse-Transkriptase-Enzym und/oder Mischungen von Nukleosidtriphosphaten, die zur Extension der Primer durch templatabhängige DNA-Synthese geeignet sind.
  3. Kit nach einem der Ansprüche 1 bis 2, ferner umfassend Reagenzien zum Ausführen von nachfolgenden Assays.
  4. Kit nach Anspruch 3, wobei die Reagenzien zum Ausführen von nachfolgenden Assays Oligonukleotidsonden für den Nachweis oder die Analyse von Einzelnukleotid-Polymorphismus, Oligonukleotid-Mikroarrays und/oder mit Schwanz versehenen Primer zur universellen Amplifikation, zum Nachweis und/oder zur Reinigung.
  5. Kit nach einem der Ansprüche 1 bis 4, ferner umfassend Reagenzien, die zur Ausführung mehrerer quantitativer Singleplex- oder Echtzeit-Amplifikationsreaktionen geeignet sind.
  6. Kit nach Anspruch 5, wobei die Reagenzien, die zur Ausführung mehrerer mehrerer quantitativer Singleplex- oder Echtzeit-Amplifikationsreaktionen geeignet sind, ein Satz quantitativer oder Echtzeit-Amplifikationsprimer, eine Oligonukleotidsonde, die mit einem Kennzeichnungssystem gekennzeichnet ist, das zur Überwachung der quantitativen Echtzeit-Amplifikationsreaktion geeignet ist, eine DNA-Polymerase bei einer Konzentration, die für eine Simplex-Amplifikationsreaktion geeignet ist, und/oder Mischungen von Desoxy-Nukleosidtriphosphaten sind, die für die templatabhängige DNA-Synthese geeignet sind.
  7. Kit nach einem der Ansprüche 1 bis 6, wobei die Anzahl der Amplifikationsprimersätze mindestens 100, 300, 500, 1.000, 10.000 oder 30.000 beträgt.
  8. Kit nach einem der Ansprüche 2 bis 7, wobei das DNA-Polymeraseenzym Taq oder Tth ist.
  9. Kit nach Anspruch 1, wobei die Amplifikationsprimer in den individuellen Reaktionsgefäßen unabhängig voneinander die gleichen wie oder verschieden von dem Satz von Primern sein können, der die mehreren Multiplex-Amplifikationsprimer umfasst.
  10. Kit nach einem der Ansprüche 1 bis 9, wobei die mehreren Reaktionsgefäße eine Multiwell-Platte ist.
  11. Kit nach einem der Ansprüche 1 bis 10, ferner umfassend eine 5'-Exonuklease-Sonde.
  12. Kit nach einem der Ansprüche 1 bis 11, wobei die Menge jedes Primers in den Amplifikationsprimersätzen nach Anspruch 1 zur Ausführung der Multiplex-Reaktion bei einer Konzentration von 30, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800 oder 900 nM pro Primer geeignet ist.
  13. Kit nach einem der Ansprüche 1 bis 12, wobei die Menge jedes Primers in den Amplifikationsprimersätzen nach Anspruch 1 zur Ausführung der Multiplex-Reaktion bei einer Konzentration von 30 nM bis 100 nM pro Primer geeignet ist.
  14. Kit nach Anspruch 2 bis 13, wobei die Reverse-Transkriptase eine AMV-Reverse-Transkriptase MuLV oder Tth - Reverse-Transkriptase ist.
  15. Multiplex-Amplifikationszusammensetzung, umfassend mehrere Amplifikationsprimersätze, wobei jeder zum Amplifizieren einer anderen Sequenz von Interesse geeignet ist, und mehrere einzelsträngige Oligonukleotidsonden, von denen jede zu allen oder einem Teil mindestens einer der Sequenzen von Interesse geeignet ist.
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